基于FGF23-Klotho轴探讨肾元颗粒对db/db糖尿病肾病小鼠骨骼的保护作用  

Study on the Protective Effect of the Bone of db/db Diabetic Nephropathy Mice with Shenyuan Granule Based on FGF23-Klotho Axis

作　　者：朱国双 王岚 邹新蓉[2,3] 吴文静[2,3] 孙龙 邓丹芳[1] 王小琴 Zhu Guoshuang;Wang Lan;Zou Xinrong;Wu Wenjing;Sun Long;Deng Danfang;Wang Xiaoqin(College of Traditional Chinese Medicine,Hubei University of Chinese Medicine,Wuhan 430061,China;Hubei Provincial Hospital of Traditional Chinese Medicine,Wuhan 430061,China;Hubei Academy of Traditional Chinese Medicine,Wuhan 430061,China)

机构地区：[1]湖北中医药大学中医临床学院,武汉430061 [2]湖北省中医院,武汉430061 [3]湖北省中医药研究院,武汉430061

出　　处：《世界科学技术-中医药现代化》2020年第1期77-83,共7页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会面上项目(81573907):基于Klotho基因/FGF23/Egr1信号通路研究温补脾肾通腑泄浊法对糖尿病肾病钙磷代谢的影响,负责人:王小琴;国家自然科学基金委员会面上项目(81874439):基于Klotho调控的FGF23/FGFR4/PLCγ信号通路研究糖尿病肾病血管钙化机制及中药的干预作用,负责人:王小琴;国家自然科学基金委员会青年项目(81703991):基于Klotho调控的FGF23/Pit1信号通路研究健脾补肾活血化浊中药对糖尿病肾病血管钙化的干预机制,负责人:王岚。

摘　　要：目的观察肾元颗粒对db/db糖尿病肾病小鼠FGF23-Klotho轴的影响,并探讨其对骨骼保护的可能机制。方法 30只db/db小鼠随机分为模型组(20 mL·kg-1)、肾元颗粒组(6.0 g·kg-1)和厄贝沙坦组(30mg·kg-1),10只同窝野生(wild type,WT)小鼠作为空白组(20 mL·kg-1),各组小鼠给药容量均为(20 mL·kg-1)。连续灌胃12周后,采用生化检测仪检测血钙、血磷含量;酶联免疫吸附测定法(ELISA)检测各组血清成纤维化生长因子23(Fibroblast growth factor 23,FGF23)含量,以及骨骼钙盐含量、骨骼碱性磷酸酶(ALP)活性;蛋白免疫印迹法(Western blot)检测骨骼骨保护素(osteoprotegerin,OPG)蛋白表达水平;免疫荧光双标检测肾脏Klotho和成纤维化生长因子受体1(Fibroblast growth factor receptor 1,FGFR1)蛋白,并于激光共聚焦下观察拍照。结果与正常组比,模型组小鼠血钙减少,血磷含量升高(P <0.05),小鼠血FGF23含量显著升高(P <0.05),小鼠骨骼中钙盐含量减少,ALP活性降低(P <0.05),小鼠骨骼OPG蛋白表达减少(P <0.05),小鼠肾脏Klotho和FGFR1免疫荧光双标阳性细胞数显著减少(P <0.05);与模型组比,肾元颗粒组小鼠血钙升高,血磷减低(P <0.05),血FGF23含量降低(P <0.05),骨骼钙盐含量升高,骨骼ALP活性升高(P <0.05),骨骼OPG蛋白表达量增加(P <0.05),肾脏免疫荧光双标阳性细胞数增多(P <0.05)。结论肾元颗粒能改善db/db糖尿病肾病小鼠骨质的流失,而其作用机制可能与FGF23-Klotho轴平衡恢复有关。Objective To observe the effect of Shenyuan Granule on the FGF23-Klotho axis of db/db diabetic nephropathy mice,and to explore the possible mechanism of bone protection.Methods Thirty db/db mice were randomly divided into a model group(20 mL·kg-1),a kidney granule group(6.0 g·kg-1),and an irbesartan group(30 mg·kg-1).Ten wild type(WT)mice were used as a blank group(20 mL·kg-1),and the dose capacity of each group was(20 mL·kg-1).After 12 weeks of continuous gavage,the levels of serum calcium and serum phosphorus were detected by biochemical detector.Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of fibroblast growth factor 23(FGF23),bone calcium content and bone alkaline phosphatase(ALP)activity in each group.Western blotting(WB)was used to detect the expression of osteoprotegerin(OPG)protein;the immunofluorescence double label was used to detect the protein of Klotho and Fibroblast growth factor receptor 1(FGFR1),and observe under laser confocal microscopy.Results Compared with the normal group,the serum calcium in the model group decreased,and the blood phosphorus content increased(P<0.05).The serum FGF23 content increased significantly(P<0.05),and the calcium content,the ALP activity decreased.The expression level of OPG protein decreased(P<0.05),and the number of immunofluorescence double-labeled positive cells in mouse kidney Klotho and FGFR1 was significantly decreased(P<0.05).Compared with the model group,the serum calcium and serum phosphorus decreased in the Shenyuan Granule group(P<0.05),and the serum FGF23 content decreased(P<0.05).The bone calcium content and the bone ALP activity increased(P<0.05).The expression of OPG protein in bone increased(P<0.05),and the number of positive immunofluorescence double-labeled cells increased(P<0.05).Conclusion Shenyuan Granule can improve bone loss in db/db diabetic nephropathy mice,and its mechanism may be related to the recovery of FGF23-Klotho axis balance.

关 键 词：钙磷代谢 肾元颗粒 糖尿病肾病 KLOTHO 成纤维化生长因子23 

分 类 号：R2-031[医药卫生—中西医结合]